7/31/14 Target: Confirm that we extracted pfu polymerase by running a PAGE gel.
(insert picture)
Our gel did not show the bands that we expected when we referenced previous successful gels. There are a variety of components that could have affected the outcome, the primary one being that the pfu polymerase was not present in the elutions. With more time, we could rerun PCR and, if needed, remake pfu polymerase. In terms of long time storage (longer than 2 days), the protocol suggests a 20% glycerol stock (compared to our conventional 50% glycerol) and then put in -20C. Once again, a PCR of samples stored for longer than 2 days would be needed to test which form works better. 7/30/14 Target: Collect a purified Pfu sample through Nickel purification.
We followed the previous protocol for centrifuging and purifying the pfu polymerase. We then nanodropped the samples.
(insert nanodrops here) 7/29/14 Target: Day 2 of pfu polymerase.
We took out the cultures we made yesterday and measured the absorbancy level. The process which took 4 hours last time only took 1.25 hours this time, which raised concern, but was attributed to our selected colonies hitting exponential growth quickly. With our extra time, we finished uploading sources to EndNote.
We got our PAGE gel back from last week. The lanes showed identical bands, which could be explained by the pfu protein breaking or contamination.
Figure 1. Our PAGE gel. Lane 1 - stored in 4C Lane 2 - stored in -20C, .5 glycerol stock
7/28/14 Target: Redo pfu, make PAGE gel for testing later, and analyze the sequence of the pfu polymerase from before.
Pfu sequence with Ns cut out. CNTTCGGGCTTTGTTAGCAGCCGGATCCTCGAGCTAGGATTTTTTAATGTTAAGCCAGGAAGTTAGGCCGACTTGTCTTGTCTTTTGGTATCTGAGGTCTTCCTTTCTGTATCCAAATC CCTCCAATATCCTAAGTACCGCTGGAAGAACCTGGTTCTCAATGTAATATTCTGCGTCATACTTGTGCTTTTTGGGATCGTATTCCTCAGCTAGAATTGCCCTATTGCTAATTGGACCA TCGCCTCTAAGTACTATGTATCCAATTACCATTCCTGGCTTTATTTTAACTCCTTTAGCAGCTAGTTTCTTTGCAGCAGCTACGTGAGGACCTATCGCCTTATACTCATGTAATGGTCTTGTTATCTGCTCATATATTGCGAGCTTCTCTGGTGGAATTTCATAATTGGCAAGCTTTTGTATTACTTCTTTTACTATTCTCACAGCTTCTTCAACATCTCCGTGTTTTAGTATTGTCT CCAAAACTCTAGCTTGAGTTTCTTTTGCAATTTCACTCCAATCTCTCCTAACTATCTCTAAACCACGAGTAATGACTTTTCCTTCTTCATCTATTACTGCATACCTCTTCTTCGTAACG AAGAATCCCCTCTTATAAAACCCTTCATATTCAAGCTCTAGCAGTCCAGGGAGCTTTGAATTTATGTATTTTACAAATTCTAGAGCCTTTATCTTTATTTCCTCACTTTCTCCTCCTGG GNTAGTTGCATAGAGACCATCAGTGTCAATGTAGAGGACTTTAAATCCAAGCTTTTCTTCGAGCTCCTTCCATACTAACTCGATGTACTTTCTTCCCCNNNAGTAACGCTCTCAGCACA CTCCTTACAGTACCATCTTGCTTTTGCATAGCCATAATATCCGTAGAAAGAATTTGCTAANAGTTTTATCGCTTTTTNNCNATAGTCANGNAGNATTTTTNCNATNNNNTCTNNNNTTC NNNCATNTTTNNNNNA
7/25/14 Target: Run PAGE gel to see if the pfu polymerase was degraded. 7/24/14 Target: Make PAGE gel.
We remade the gel twice today, because the first time the 12% bottom layer did not polymerize appropriately. We finished the second set and stored it away to test tomorrow.
7/23/14 Target: Make PAGE gel to see if the pfu polymerase had degraded.
We began to make the PAGE gel to analyze the degradation of the pfu. Structurally, the PAGE gel is different from the regular gels we make in that it is vertical and requires two separate layers.
(insert pic)
The top layer was not dyed and the wells that the comb had created could not be seen when submerged in the TGS buffer. 7/22/14 Target: Redo PCR from yesterday.
We redid the PCR from yesterday. We triple-checked the gel and placed the samples in the wells. We also allowed another researcher to borrow a lane. After running the gel, we found that there was nothing for any of our samples. Our ladders and the other researchers bands had appeared, meaning the gel was not comprised. Confusion arose because we had proof the the pfu polymerase had worked last week, and we became concern that the pfu polymerase had degraded. We tested the concentrations in the nanodrop and found a stunning rise in concentration (insert pictures here). This rise can be attested to the evaporation of the buffer that the polymerase was in, but didn't prove/disprove the quality of the polymerase. We resolved to test the protein with a PAGE gel.
7/21/14 Target: Create oligo-mix for cloning PfGr/DhFr and run PCR to determine the optimal storage conditions of pfu polymerase.
A. Making an Oligo-Mix and Unexpected Breath Holding
Because of last week's success with the pfu, we moved on to cloning. We were assigned two genes - one for DHFR and one for PFGR. Attempts to clones these genes had already been made, but due to its size, the attempts were not successful. We teamed up with BraxtonS. and SarahH. taking DHFR and MartinK and I taking PGFR. We began by checking the methods of previous methods. Outlined in the document were the primers (38 for each) and other information regarding each of our genes. We then began making the two oligo-mixes. We took measures to maintain the future integrity of the wells by taking precautions like covering extra wells with sterile chem-wipes and not breathing/talking directly over the box. We then stored the two mixes away to be used for PCRs later.
B. PCR
When we left last week, we stored away the pfu polymerase in 4C and another sample in glycerol in the -20C freezer. We also stored away the second elution (of lower concentration) of the pfu from the Ni-NTA purification. We ran a PCR maintaining the same cycles that previously worked on the three samples to test which storage condition worked the best and whether the second elution presented as viable source of polymerase.
After running the PCR, we looked at the gel underneath the UV light and the only thing we could see was a bright orange smudge. After considerable confusion, we had to figure out what went wrong. We ruled out the lack of EtBr (as there was the smudge) and figured out that if even the ladder hadn't shown up, there had to be something wrong with the gel. And indeed, it was discovered that the 1% agarose gel was made out of water instead of TAE.
Week 7
Great work Roshni! You guys did an impressive job of getting the competent cells to work and the Pfu Polymerase. Good job on looking up the conditions necessary for PCR of PFU poly. - Dr. B 072114
Can you list the exact temps/times used for Pfu polymerase? I am confused if that is the denaturation temp or the extension... etc.
7/18/14 Target: Verify denaturation temperature of pfu polymerase.
Using the gel from yesterday, we determined that the pfu worked best in 98C and 100C.
We ran a PCR today with temperature as the independent variable.
(insert picture here)
Our gel showed that the 98C worked the best, as it it showed the same light intensity progression in the increasing concentrations as seen with the control Taq polymerase gel.
After we determined that the pfu worked, we stored a fourth of the elution in a 50% glycerol stock. The remaining was placed back in the 4C freezer. The conditions of storage will be another variable to explore in later procedures (cloning). Results: Therefore, the cycle that worked for Pfu polymerase (on PNIC-bsa4 with PLIC-for/rev) 95C for 30 sec 98C for 5s 55.7C for 30 sec 72C for 1 min 72C for 2.5 min 4C for infinity (we did 30 cycles) 7/17/14 Target: Determine the appropriate PCR conditions for the pfu polymerase.
Yesterday's gel showed us that the PCR conditions described for Taq polymerase were not effective for PCR. Today's first challenge was to determine the optimal conditions for pfu polymerase.
In the spirit of discovery, we decided to do some online research and use publications of papers which had used pfu. We found a variety of results, but we had to consider alternate variable that could affect the temperatures of the phases. We had to make sure that our optimal pfu temperature didn't negatively effect the primers or other materials.
We also decided to reference the protocol used for Q5, another polymerase. Using the information we had gathered from both sources, we determined that the appropriate range to test in would be 92-100C.
To test the range, we conducted a temperature gradient, putting the 8 samples in one degree increments.
(insert picture here)
Our gel showed two prominent bands - one in 98C and one in 100C. This gel also explains why the pfu polymerase didn't work yesterday - the temperatures simply did not the meet the requirements of the polymerase.
(insert gel pic here)
7/16/14 Target: Compare the PCR results of taq and pfu on pNIC-bsa4.
Today, we ran two separate PCRs - one with taq polymerase and the other with pfu. We used the same concept (three increasing concentrations) as the PCR we did on PGBR22 because we would want our gel to resemble (in terms of light intensity progression) the previous one.
We used pLIC for and rev primers, pNIC template, and the temperatures prescribed for taq to see how the pfu would work (if it would at all).
Our challenge today was to determine the appropriate annealing temperature for the pLIC for and rev primers. We used the IDT database to find the melting temperatures of the primers and determined 56.7C as an appropriate temperature (averaged out and dropped 5 degrees to make sure not to melt the primers).
While the annealing probably (hopefully) worked well, our gel was not deemed successful. Our results were not unexpected because different polymerases are optimized to work at different temperatures. (insert gel pic and a wonderful caption here)
7/15/14 Target: Calcium Competent Cells transformation efficiency. The Day of Truth
We tested the Calcium Competent Cells by transforming them.
Figure 1. 1ng DNA, 15 ul bacteria. We can see 3 colonies on the plate.
Figure 2. 5ng of DNA and 15 ul of bacteria. ~130 colonies.
Figure 3. Here we had 25 ng of DNA and 15 ul of bacteria. ~300 colonies.
The colonies indicated the competency of the cells. 7/14/14 Target: Our first PCR!
After the purification of the pfu polymerase, the next step was to begin testing if the pfu worked in PCR.
Before begining the process, we first had to practice the protocol for PCR and understand what was actually happening when we did PCR.
LuisV. drew us a wonderful diagram explaining the the three steps of PCR (Denaturation, Annealing, Elongation) and taught us the functionalities of the different items we would be putting together (for example, what dNTPs meant).
We used PGBR22 and taq polymerase for our practice because the protocol and results were well known, so we could identify if we had done the procedure correctly.
(insert gel here)
We had a bit of a plateau in the intensity of the light in our 3 and 30 concentrations which could be cause by the exhaustion of dNTPs or primers. We deemed the gel mostly successful because we had a increase in intensity between the .3 and 3 concentrations.
Roshni - ok good work. You don't ahve to have as much procedural detail - that can go in your labnotebook more. You want to focus on images of results here and brief analysis. That is kind of hard for this latest protocol because you have been making something - but don't really have a results image yet - so that is ok. -- Dr. B
Week 6
7/11/14
Tacc Research Lab 7/10/14 Target: Calcium Competent Cells Redux
- An early day began by adding our starter culture to 1 L of LB and measuring the OD every hour.
Figure 1. This is the accumulation of the OD600 measurements. Each of the lines show the concentration increasing. Measurements were taken every hour.
- After the culture reached .35 we added in our buffers that we previously made.
Figure 2. Our buffers from before. These buffers were added to resuspend the cells after the centrifuge rendered the cells a pellet. They helped concentrate the cells.
7/9/14 Target: Midi-prep and Calcium Competent Cells.
(otherwise known as Triumph and Tragedy)
A. The Quest to Figure Out What the Lab Gave Us (Triumph) - SarahH and MartinK began the process of Midi-Prepping the pfu polymerase. After the results were NanoDropped.
Figure 1. Our Midi-Prep produced a viable concentration for sequencing.
B. Calcium Competent Cells
- BraxtonS. and I began the Calcium Competent Cells by creating the flasks and checking their concentration hourly at OD600. The concentrations were promising and growing at the exponential rate that is expected. As the day wound to an end we were unfortunately informed that the protocol was an 8 hour process and that the growths we had done that day would have to be done again. Though a slight bit disappointing, repeating the procedure the next day would give us the opportunity to practice the protocol and perfect any errors we might have made today. - We redid the starter culture of cells from Day 2 and stored them in an incubator overnight. We also discarded and washed the other flasks. 7/8/14 Target: Another multi-protocol day!
A. Day 2 of glycerol stocks stored in cryovials tubes.
B.Calcium Competent Cells
- made buffers for the Ca competent cells (and then autoclaved)
- prepared starter culture of LB+ DH5a colony to grow overnight
C. The Quest to Figure Out What the Lab Gave Us
- Verification is understandably important in the lab because the smallest error could corrupt weeks worth of research or create a safety hazard. So it only makes sense, then, that we verified the pfu polymerase that we obtained from another lab. We began the process today by putting the pfu into two flasks (with one for backup) and left them overnight. We will midi-prep them later and then send them off to sequencing. There is a slight concern about the concentration because the cells are BL21 which are made to analyze protein, not DNA. It is suspense that must wait till sequencing.To be continued. 7/7/14 Target: A multi-protocol day.
A. Everything Must Go! (Except the pfu) - continued the pfu protein purification from 7/3 using Ni-NTA resin.
Figure 1. This is the column containing the Ni-NTA resin (the thin layer of bluish substance at the bottom). The pfu hooks onto the Ni while the other proteins flow through the column. The pfu protein is then flushed out with Buffers (ours contained imidiazole).
- We took samples throughout and ended up with two elutions at end. We nanodropped the elutions to check for the concentration of purification.
Figure 2. The first elution from the purification nano-drop results.
Figure 3. The second elution from the purification nano-drop results.
Figure 4. The second elution from the purification nano-drop results. This test was for verification.
B. Calcium Competent Cells - The Origins
- Made LB plates with DH5a (made to analyze DNA) without amp to begin Calcium Competent Cells
Figure 2. These are the plates with DH5a. They are appropriately labeled to indicated contents. The colonies that grow on these plates will later be harvested for transformation.
C. Day 1 of glycerol stocks. These were stored to be used later in the -80C freezer.
Week 5
7/4/14
Fourth of July (Holiday) 7/3/14 Target: Continue the Pfu purification process (1. Make buffers 2. Spin down.)
- overnight incubation of both flasks resulted in cloudy liquid in both flasks indicating growth
- Flask S was taken to to be spun-down in centrifuge using a JA10 rotor for 500 ml (Flask B had no IPTG, and therefore will be treated later)
- the solution was in the centrifuge for 20 min at 4C at 7000 RPM
Figure 1. The pellet resulting from the force of the centrifuge.
- the resulting pellet was resuspended in 12 ml of Buffer A
- the solution was then sonicated to lyse the cells
Figure 2. Our pfu solution prepared and ready to by lysed in the sonicator.
the lysate was centrifuged in a JA10 rotor
Figure 3. Due to a larger quantity of material that needed to be spun down, a larger centrifuge (pictured here) was used.
7/2/14 Target: Further steps for Pfu purification.
- made sets of 500 ml of LB +amp+ overnight culture into each of two 2 Liter flasks - referred to as Flask S and B - (as the size of the flask must be four times the volume of the solution)
- incubated the two flasks (Flask S and Flask B) in the 37C shaking incubator for 30 min.
- tested concentration at OD600 - both flasks had a concentration of 0.0 for two hours (checked every 30 minutes).
- after the incubation period of two hours Flask S rose to the concentration of .012 while Flask B showed 0.0
- after incubation of another two hours, IPTG was added to Flask S
- both flasks (S and B) were incubated overnight at 37 C
7/1/14 Target: Complete gel procedure from yesterday and begin protocol for Pfu purification.
- calculated concentrations of solutions in wells a) uncut plasmid, b) 3 different cuts (EcoRI, pvuII, and EcoRI + pvuII)
- made gel
- tested the samples in gel
- negative control - uncut plasmid
Figure 1. Gel from restriction enzyme digest. 1% Agarose gel Lane 1 - Empty lane Lane 2 - 1kb ladder Lane 3 - Caroline uncut plasmid Lane 4- C EcoRI Lane 5 - C pvuII Lane 6 - C EcoRI+pvuII lane 7 - Roshni uncut plasmid Lane 8 - R EcoRI Lane 9 - R pvuII Lane 10 - EcoRI+ pvuII
- the lower bands are dimmer because of a lower concentration of Ethidium Bromide.
Pfu Purification
- began purification process by adding amp to two agar plates
- incubated the samples overnight at 37 C
6/30/14 Target: Analyze the second set of PGBR22 samples using a restriction enzyme digest and visualize the fragmentation in gel.
- calculated the various concentrations and amounts of the plasmid, enzyme buffers, restriction enzyme, and DDW.
- three different preparations of reactions: EcoRI, pvuII, and EcoRI + pvuII
- Negative control* : Regular PGBR22 plasmid without restriction enzymes
- Made gel for the gel electrophoresis
- gel ultimately not tested due to time constraints
- enzyme stop process followed to ensure
Week 4
6/27/14 -Target: Test the second sample of PGBR22 using gel electrophoresis and DNA sequencing.
- Sent second set of samples off to sequencing.
- prepared 1% Agarose Gel to see the bands and also to see the level of degradation given the origin of the samples from the backup set of flasks.
- Results: 6/26/14 - Target: Conduct a restriction enzyme digest testing the PGBR22 plasmids.
- Achieved: Due to the samples being left outside overnight, a second midi-prep was done the samples from which are hereby defined as samples of the second set.
- Ran a gel to test the samples of PGBR22 from set one using the concentration of 293.3 ng/ul. This was to test for the potential of degradation, given that the samples in the were plasmids and not proteins, they stood less chance of denaturing given the environment. Results: The gel showed degradation, but less so than expected.
6/25/14
- Target: Send of samples of PGBR22 to sequencing and make LB and agar plates.
- 6/24/14 Target: Conducted a midi-prep to isolate DNA from PGBR22.
-after completing the midi-prep, a Nano-Drop was conducted to test the concentration of the extraction in ng/ul. 6/23/14 Target: Understand the process of analyzing a DNA sequences and restriction enzymes by using computer programs.
- prepare flasks of LB to process the next day.
Week 9
7/31/14
Target: Confirm that we extracted pfu polymerase by running a PAGE gel.
(insert picture)
Our gel did not show the bands that we expected when we referenced previous successful gels. There are a variety of components that could have affected the outcome, the primary one being that the pfu polymerase was not present in the elutions. With more time, we could rerun PCR and, if needed, remake pfu polymerase. In terms of long time storage (longer than 2 days), the protocol suggests a 20% glycerol stock (compared to our conventional 50% glycerol) and then put in -20C. Once again, a PCR of samples stored for longer than 2 days would be needed to test which form works better.
7/30/14
Target: Collect a purified Pfu sample through Nickel purification.
We followed the previous protocol for centrifuging and purifying the pfu polymerase. We then nanodropped the samples.
(insert nanodrops here)
7/29/14
Target: Day 2 of pfu polymerase.
We took out the cultures we made yesterday and measured the absorbancy level. The process which took 4 hours last time only took 1.25 hours this time, which raised concern, but was attributed to our selected colonies hitting exponential growth quickly. With our extra time, we finished uploading sources to EndNote.
We got our PAGE gel back from last week. The lanes showed identical bands, which could be explained by the pfu protein breaking or contamination.
Target: Redo pfu, make PAGE gel for testing later, and analyze the sequence of the pfu polymerase from before.
Pfu sequence with Ns cut out.
CNTTCGGGCTTTGTTAGCAGCCGGATCCTCGAGCTAGGATTTTTTAATGTTAAGCCAGGAAGTTAGGCCGACTTGTCTTGTCTTTTGGTATCTGAGGTCTTCCTTTCTGTATCCAAATC
CCTCCAATATCCTAAGTACCGCTGGAAGAACCTGGTTCTCAATGTAATATTCTGCGTCATACTTGTGCTTTTTGGGATCGTATTCCTCAGCTAGAATTGCCCTATTGCTAATTGGACCA
TCGCCTCTAAGTACTATGTATCCAATTACCATTCCTGGCTTTATTTTAACTCCTTTAGCAGCTAGTTTCTTTGCAGCAGCTACGTGAGGACCTATCGCCTTATACTCATGTAATGGTCTTGTTATCTGCTCATATATTGCGAGCTTCTCTGGTGGAATTTCATAATTGGCAAGCTTTTGTATTACTTCTTTTACTATTCTCACAGCTTCTTCAACATCTCCGTGTTTTAGTATTGTCT
CCAAAACTCTAGCTTGAGTTTCTTTTGCAATTTCACTCCAATCTCTCCTAACTATCTCTAAACCACGAGTAATGACTTTTCCTTCTTCATCTATTACTGCATACCTCTTCTTCGTAACG
AAGAATCCCCTCTTATAAAACCCTTCATATTCAAGCTCTAGCAGTCCAGGGAGCTTTGAATTTATGTATTTTACAAATTCTAGAGCCTTTATCTTTATTTCCTCACTTTCTCCTCCTGG
GNTAGTTGCATAGAGACCATCAGTGTCAATGTAGAGGACTTTAAATCCAAGCTTTTCTTCGAGCTCCTTCCATACTAACTCGATGTACTTTCTTCCCCNNNAGTAACGCTCTCAGCACA
CTCCTTACAGTACCATCTTGCTTTTGCATAGCCATAATATCCGTAGAAAGAATTTGCTAANAGTTTTATCGCTTTTTNNCNATAGTCANGNAGNATTTTTNCNATNNNNTCTNNNNTTC
NNNCATNTTTNNNNNA
Reverse Complement
TNNNNNAAANATGNNNGAANNNNAGANNNNATNGNAAAAATNCTNCNTGACTATNGNNAA
AAAGCGATAAAACTNTTAGCAAATTCTTTCTACGGATATTATGGCTATGCAAAAGCAAGA
TGGTACTGTAAGGAGTGTGCTGAGAGCGTTACTNNNGGGGAAGAAAGTACATCGAGTTAG
TATGGAAGGAGCTCGAAGAAAAGCTTGGATTTAAAGTCCTCTACATTGACACTGATGGTC
TCTATGCAACTANCCCAGGAGGAGAAAGTGAGGAAATAAAGATAAAGGCTCTAGAATTTG
TAAAATACATAAATTCAAAGCTCCCTGGACTGCTAGAGCTTGAATATGAAGGGTTTTATA
AGAGGGGATTCTTCGTTACGAAGAAGAGGTATGCAGTAATAGATGAAGAAGGAAAAGTCA
TTACTCGTGGTTTAGAGATAGTTAGGAGAGATTGGAGTGAAATTGCAAAAGAAACTCAAG
CTAGAGTTTTGGAGACAATACTAAAACACGGAGATGTTGAAGAAGCTGTGAGAATAGTAA
AAGAAGTAATACAAAAGCTTGCCAATTATGAAATTCCACCAGAGAAGCTCGCAATATATG
AGCAGATAACAAGACCATTACATGAGTATAAGGCGATAGGTCCTCACGTAGCTGCTGCAA
AGAAACTAGCTGCTAAAGGAGTTAAAATAAAGCCAGGAATGGTAATTGGATACATAGTAC
TTAGAGGCGATGGTCCAATTAGCAATAGGGCAATTCTAGCTGAGGAATACGATCCCAAAA
AGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTTCTTCCAGCGGTACTTAGGA
TATTGGAGGGATTTGGATACAGAAAGGAAGACCTCAGATACCAAAAGACAAGACAAGTCG
GCCTAACTTCCTGGCTTAACATTAAAAAATCCTAGCTCGAGGATCCGGCTGCTAACAAAG
CCCGAANG
Translate results
>rf 1 Untitled reverse complement
XXKXXXXXXXXXKXLXDYXXKAIKLLANSFYGYYGYAKARWYCKECAESVTXGEESTSS*
YGRSSKKSLDLKSSTLTLMVSMQLXQEEKVRK*R*RL*NL*NT*IQSSLDC*SLNMKGFI
RGDSSLRRRGMQMKKEKSLLVV*R*LGEIGVKLQKKLKLEFWRQY*NTEMLKKL*E
KK*YKSLPIMKFHQRSSQYMSR*QDHYMSIRR*VLT*LLQRN*LLKELK*SQEW*LDT*Y
LEAMVQLAIGQF*LRNTIPKSTSMTQNITLRTRFFQRYLGYWRDLDTERKTSDTKRQDKS
A*LPGLTLKNPSSRIRLLTKPE
Translate results
>rf 2 Untitled reverse complement
XXXMXEXXXXXXKXXXTXXKKR*NX*QILSTDIMAMQKQDGTVRSVLRALLXGKKVHRVS
MEGARRKAWI*SPLH*H*WSLCNXPRRRK*GNKDKGSRICKIHKFKAPWTARA*I*RVL*
EGILRYEEEVCSNR*RRKSHYSWFRDS*ERLE*NCKRNSS*SFGDNTKTRRC*RSCENSK
RSNTKACQL*NSTREARNI*ADNKTIT*V*GDRSSRSCCKETSC*RS*NKARNGNWIHST
*RRWSN*Q*GNSS*GIRSQKAQV*RRILH*EPGSSSGT*DIGGIWIQKGRPQIPKDKTSR
PNFLA*H*KILARGSGC*QSPX
Translate results
>rf 3 Untitled reverse complement
XXXXXXXRXXXKNXX*LXXKSDKTXSKFFLRILWLCKSKMVL*GVC*ERYXXGRKYIELV
WKELEEKLGFKVLYIDTDGLYATXPGGESEEIKIKALEFVKYINSKLPGLLELEYEGFYK
RGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVK
EVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAAAKKLAAKGVKIKPGMVIGYIVL
RGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVG
LTSWLNIKKS*LEDPAANKARX
DNA polymerase [Pyrococcus furiosus]
Sequence ID: ref|WP_011011325.1|Length: 775Number of Matches: 1
See 9 more title(s)
Query 6 GRKYIELVWKELEEKLGFKVLYIDTDGLYATXPGGESEEIKIKALEFVKYINSKLPGLLE 65
GRKYIELVWKELEEK GFKVLYIDTDGLYAT PGGESEEIK KALEFVKYINSKLPGLLE
Sbjct 518 GRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLE 577
Query 66 LEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDV 125
LEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDV
Sbjct 578 LEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDV 637
Query 126 EEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAAAKKLAAKGVKIKPG 185
EEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVA AKKLAAKGVKIKPG
Sbjct 638 EEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPG 697
Query 186 MVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLR 245
MVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLR
Sbjct 698 MVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLR 757
Query 246 YQKTRQVGLTSWLNIKKS 263
YQKTRQVGLTSWLNIKKS
Sbjct 758 YQKTRQVGLTSWLNIKKS 775
These are the results for the PFU forward samples:
Revised Version with ‘N’s Cut Off
ATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGTCATATGATTTTAGATGTGGATTACATAA
CTGAAGAAGGAAAACCTGTTATTAGGCTATTCAAAAAAGAGAACGGAAAATTTAAGATAGAGCATGATAGAACTTTTAGACCATACATTTACGCTCTTCTCAGGGATGATTCAAAGATT
GAAGAAGTTAAGAAAATAACGGGGGAAAGGCATGGAAAGATTGTGAGAATTGTTGATGTAGAGAAGGTTGAGAAAAAGTTTCTCGGCAAGCCTATTACCGTGTGGAAACTTTATTTGGA
ACATCCCCAAGATGTTCCCACTATTAGAGAAAAAGTTAGAGAACATCCAGCAGTTGTGGACATCTTCGAATACGATATTCCATTTGCAAAGAGATACCTCATCGACAAAGGCCTAATAC
CAATGGAGGGGGAAGAAGAGCTAAAGATTCTTGCCTTCGATATAGAAACCCTCTATCACGAAGGAGAAGAGTTTGGAAAAGGCCCAATTATAATGATTAGTTATGCAGATGAAAATGA
AGCAAAGGTGATTACTTGGAAAAACATAGATCTTCCATACGTTGAGGTTGTATCAAGCGAGAGAGAGATGATAAAGAGATTTCTCAGGATTATCAGGGAGAAGGATCCTGACATTATAG
TTACTTATAATGGAGACTCATTCGACTTCCCACATTTAGCGAAAAGGGCAGAAAAACTTGGGATTAAATTAACCATTGGAAGAGATGGAAGCGAGCCCAAGATGCAGAGAATAGGCGAT
ATGACGGCTGTAGAAGTCANGGGAAGAATACATTTCGACTTGTATCATGTAATAACAAGGACAATAAATCTCCCAANATACACACTAGAGGCTGTATATGAANCAATTTTTGGAAAGCC
NA
Translate results
>rf 1 Untitled
ILFNFKKEIYHGPSSSSSSSSSQQRPYRRSSYDFRCGLHN*RRKTCY*AIQKRERKI*DR
ANF*TIHLRSSQG*FKD*RS*ENNGGKAWKDCENC*CREG*EKVSRQAYYRVETLFGT
SPRCSHY*RKS*RTSSSCGHLRIRYSICKEIPHRQRPNTNGGGRRAKDSCLRYRNPLSRR
RRVWKRPNYND*LCR*K*SKGDYLEKHRSSIR*GCIKRERDDKEISQDYQGEGS*HYSYL
*WRLIRLPTFSEKGRKTWD*INHWKRWKRAQDAENRRYDGCRSXGKNTFRLVSCNNKDNK
SPXIHTRGCI*XNFWKA
Translate results
>rf 2 Untitled
FCLTLRRRYTMGHHHHHHHHHHSSGHIEGRHMILDVDYITEEGKPVIRLFKKENGKFKIE
HDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEH
PQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEG
EEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTY
NGDSFDFPHLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVXGRIHFDLYHVITRTIN
LPXYTLEAVYEXIFGKP
Translate results
>rf 3 Untitled
FV*L*EGDIPWAIIIIIIIIITAAAISKVVI*F*MWIT*LKKENLLLGYSKKRTENLR*S
MIELLDHTFTLFSGMIQRLKKLRK*RGKGMERL*ELLM*RRLRKSFSASLLPCGNFIWNI
PKMFPLLEKKLENIQQLWTSSNTIFHLQRDTSSTKA*YQWRGKKS*RFLPSI*KPSITKE
KSLEKAQLLVMQMKMKQR*LLGKT*IFHTLRLYQARERRDFSGLSGRRILTL*LLI
METHSTSHI*RKGQKNLGLN*PLEEMEASPRCRE*AI*RL*KSXEEYISTCIMQGQ*I
SQXTH*RLYMXQFLESX
DNA polymerase [Pyrococcus furiosus]
Sequence ID: ref|WP_011011325.1|Length: 775Number of Matches: 1
See 9 more title(s)
Query 32 MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHG 91
MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHG
Sbjct 1 MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHG 60
Query 92 KIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY 151
KIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY
Sbjct 61 KIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY 120
Query 152 LIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPY 211
LIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPY
Sbjct 121 LIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPY 180
Query 212 VEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPHLAKRAEKLGIKLTIGRDGSEPK 271
VEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFP+LAKRAEKLGIKLTIGRDGSEPK
Sbjct 181 VEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPK 240
Query 272 MQRIGDMTAVEVXGRIHFDLYHVITRTINLPXYTLEAVYEXIFGKP 317
MQRIGDMTAVEV GRIHFDLYHVITRTINLP YTLEAVYE IFGKP
Sbjct 241 MQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKP 286
Week 8
7/25/14
Target: Run PAGE gel to see if the pfu polymerase was degraded.
7/24/14
Target: Make PAGE gel.
We remade the gel twice today, because the first time the 12% bottom layer did not polymerize appropriately. We finished the second set and stored it away to test tomorrow.
7/23/14
Target: Make PAGE gel to see if the pfu polymerase had degraded.
We began to make the PAGE gel to analyze the degradation of the pfu. Structurally, the PAGE gel is different from the regular gels we make in that it is vertical and requires two separate layers.
(insert pic)
The top layer was not dyed and the wells that the comb had created could not be seen when submerged in the TGS buffer.
7/22/14
Target: Redo PCR from yesterday.
We redid the PCR from yesterday. We triple-checked the gel and placed the samples in the wells. We also allowed another researcher to borrow a lane. After running the gel, we found that there was nothing for any of our samples. Our ladders and the other researchers bands had appeared, meaning the gel was not comprised. Confusion arose because we had proof the the pfu polymerase had worked last week, and we became concern that the pfu polymerase had degraded. We tested the concentrations in the nanodrop and found a stunning rise in concentration (insert pictures here). This rise can be attested to the evaporation of the buffer that the polymerase was in, but didn't prove/disprove the quality of the polymerase. We resolved to test the protein with a PAGE gel.
7/21/14
Target: Create oligo-mix for cloning PfGr/DhFr and run PCR to determine the optimal storage conditions of pfu polymerase.
A. Making an Oligo-Mix and Unexpected Breath Holding
Because of last week's success with the pfu, we moved on to cloning. We were assigned two genes - one for DHFR and one for PFGR. Attempts to clones these genes had already been made, but due to its size, the attempts were not successful. We teamed up with BraxtonS. and SarahH. taking DHFR and MartinK and I taking PGFR. We began by checking the methods of previous methods. Outlined in the document were the primers (38 for each) and other information regarding each of our genes. We then began making the two oligo-mixes. We took measures to maintain the future integrity of the wells by taking precautions like covering extra wells with sterile chem-wipes and not breathing/talking directly over the box. We then stored the two mixes away to be used for PCRs later.
B. PCR
When we left last week, we stored away the pfu polymerase in 4C and another sample in glycerol in the -20C freezer. We also stored away the second elution (of lower concentration) of the pfu from the Ni-NTA purification. We ran a PCR maintaining the same cycles that previously worked on the three samples to test which storage condition worked the best and whether the second elution presented as viable source of polymerase.
After running the PCR, we looked at the gel underneath the UV light and the only thing we could see was a bright orange smudge. After considerable confusion, we had to figure out what went wrong. We ruled out the lack of EtBr (as there was the smudge) and figured out that if even the ladder hadn't shown up, there had to be something wrong with the gel. And indeed, it was discovered that the 1% agarose gel was made out of water instead of TAE.
Week 7
Great work Roshni! You guys did an impressive job of getting the competent cells to work and the Pfu Polymerase. Good job on looking up the conditions necessary for PCR of PFU poly. - Dr. B 072114
Can you list the exact temps/times used for Pfu polymerase? I am confused if that is the denaturation temp or the extension... etc.
7/18/14
Target: Verify denaturation temperature of pfu polymerase.
Using the gel from yesterday, we determined that the pfu worked best in 98C and 100C.
We ran a PCR today with temperature as the independent variable.
(insert picture here)
Our gel showed that the 98C worked the best, as it it showed the same light intensity progression in the increasing concentrations as seen with the control Taq polymerase gel.
After we determined that the pfu worked, we stored a fourth of the elution in a 50% glycerol stock. The remaining was placed back in the 4C freezer. The conditions of storage will be another variable to explore in later procedures (cloning).
Results: Therefore, the cycle that worked for Pfu polymerase (on PNIC-bsa4 with PLIC-for/rev)
95C for 30 sec
98C for 5s
55.7C for 30 sec
72C for 1 min
72C for 2.5 min
4C for infinity
(we did 30 cycles)
7/17/14
Target: Determine the appropriate PCR conditions for the pfu polymerase.
Yesterday's gel showed us that the PCR conditions described for Taq polymerase were not effective for PCR. Today's first challenge was to determine the optimal conditions for pfu polymerase.
In the spirit of discovery, we decided to do some online research and use publications of papers which had used pfu. We found a variety of results, but we had to consider alternate variable that could affect the temperatures of the phases. We had to make sure that our optimal pfu temperature didn't negatively effect the primers or other materials.
We also decided to reference the protocol used for Q5, another polymerase. Using the information we had gathered from both sources, we determined that the appropriate range to test in would be 92-100C.
To test the range, we conducted a temperature gradient, putting the 8 samples in one degree increments.
(insert picture here)
Our gel showed two prominent bands - one in 98C and one in 100C. This gel also explains why the pfu polymerase didn't work yesterday - the temperatures simply did not the meet the requirements of the polymerase.
(insert gel pic here)
7/16/14
Target: Compare the PCR results of taq and pfu on pNIC-bsa4.
Today, we ran two separate PCRs - one with taq polymerase and the other with pfu. We used the same concept (three increasing concentrations) as the PCR we did on PGBR22 because we would want our gel to resemble (in terms of light intensity progression) the previous one.
We used pLIC for and rev primers, pNIC template, and the temperatures prescribed for taq to see how the pfu would work (if it would at all).
Our challenge today was to determine the appropriate annealing temperature for the pLIC for and rev primers. We used the IDT database to find the melting temperatures of the primers and determined 56.7C as an appropriate temperature (averaged out and dropped 5 degrees to make sure not to melt the primers).
While the annealing probably (hopefully) worked well, our gel was not deemed successful.
Our results were not unexpected because different polymerases are optimized to work at different temperatures.
(insert gel pic and a wonderful caption here)
7/15/14
Target: Calcium Competent Cells transformation efficiency.
The Day of Truth
We tested the Calcium Competent Cells by transforming them.
The colonies indicated the competency of the cells.
7/14/14
Target: Our first PCR!
After the purification of the pfu polymerase, the next step was to begin testing if the pfu worked in PCR.
Before begining the process, we first had to practice the protocol for PCR and understand what was actually happening when we did PCR.
LuisV. drew us a wonderful diagram explaining the the three steps of PCR (Denaturation, Annealing, Elongation) and taught us the functionalities of the different items we would be putting together (for example, what dNTPs meant).
We used PGBR22 and taq polymerase for our practice because the protocol and results were well known, so we could identify if we had done the procedure correctly.
(insert gel here)
We had a bit of a plateau in the intensity of the light in our 3 and 30 concentrations which could be cause by the exhaustion of dNTPs or primers. We deemed the gel mostly successful because we had a increase in intensity between the .3 and 3 concentrations.
Roshni - ok good work. You don't ahve to have as much procedural detail - that can go in your labnotebook more. You want to focus on images of results here and brief analysis. That is kind of hard for this latest protocol because you have been making something - but don't really have a results image yet - so that is ok. -- Dr. B
Week 6
7/11/14
Tacc Research Lab
7/10/14
Target: Calcium Competent Cells Redux
- An early day began by adding our starter culture to 1 L of LB and measuring the OD every hour.
- After the culture reached .35 we added in our buffers that we previously made.
Target: Midi-prep and Calcium Competent Cells.
(otherwise known as Triumph and Tragedy)
A. The Quest to Figure Out What the Lab Gave Us (Triumph)
- SarahH and MartinK began the process of Midi-Prepping the pfu polymerase. After the results were NanoDropped.
B. Calcium Competent Cells
- BraxtonS. and I began the Calcium Competent Cells by creating the flasks and checking their concentration hourly at OD600. The concentrations were promising and growing at the exponential rate that is expected. As the day wound to an end we were unfortunately informed that the protocol was an 8 hour process and that the growths we had done that day would have to be done again. Though a slight bit disappointing, repeating the procedure the next day would give us the opportunity to practice the protocol and perfect any errors we might have made today.
- We redid the starter culture of cells from Day 2 and stored them in an incubator overnight. We also discarded and washed the other flasks.
7/8/14
Target: Another multi-protocol day!
A. Day 2 of glycerol stocks stored in cryovials tubes.
B.Calcium Competent Cells
- made buffers for the Ca competent cells (and then autoclaved)
- prepared starter culture of LB+ DH5a colony to grow overnight
C. The Quest to Figure Out What the Lab Gave Us
- Verification is understandably important in the lab because the smallest error could corrupt weeks worth of research or create a safety hazard. So it only makes sense, then, that we verified the pfu polymerase that we obtained from another lab. We began the process today by putting the pfu into two flasks (with one for backup) and left them overnight. We will midi-prep them later and then send them off to sequencing. There is a slight concern about the concentration because the cells are BL21 which are made to analyze protein, not DNA. It is suspense that must wait till sequencing.To be continued.
7/7/14
Target: A multi-protocol day.
A. Everything Must Go! (Except the pfu)
- continued the pfu protein purification from 7/3 using Ni-NTA resin.
- We took samples throughout and ended up with two elutions at end. We nanodropped the elutions to check for the concentration of purification.
B. Calcium Competent Cells - The Origins
- Made LB plates with DH5a (made to analyze DNA) without amp to begin Calcium Competent Cells
Week 5
7/4/14
Fourth of July (Holiday)
7/3/14
Target: Continue the Pfu purification process (1. Make buffers 2. Spin down.)
- overnight incubation of both flasks resulted in cloudy liquid in both flasks indicating growth
- Flask S was taken to to be spun-down in centrifuge using a JA10 rotor for 500 ml (Flask B had no IPTG, and therefore will be treated later)
- the solution was in the centrifuge for 20 min at 4C at 7000 RPM
- the resulting pellet was resuspended in 12 ml of Buffer A
- the solution was then sonicated to lyse the cells
the lysate was centrifuged in a JA10 rotor
7/2/14
Target: Further steps for Pfu purification.
- made sets of 500 ml of LB +amp+ overnight culture into each of two 2 Liter flasks - referred to as Flask S and B - (as the size of the flask must be four times the volume of the solution)
- incubated the two flasks (Flask S and Flask B) in the 37C shaking incubator for 30 min.
- tested concentration at OD600 - both flasks had a concentration of 0.0 for two hours (checked every 30 minutes).
- after the incubation period of two hours Flask S rose to the concentration of .012 while Flask B showed 0.0
- after incubation of another two hours, IPTG was added to Flask S
- both flasks (S and B) were incubated overnight at 37 C
7/1/14
Target: Complete gel procedure from yesterday and begin protocol for Pfu purification.
- calculated concentrations of solutions in wells a) uncut plasmid, b) 3 different cuts (EcoRI, pvuII, and EcoRI + pvuII)
- made gel
- tested the samples in gel
- negative control - uncut plasmid
- the lower bands are dimmer because of a lower concentration of Ethidium Bromide.
Pfu Purification
- began purification process by adding amp to two agar plates
- incubated the samples overnight at 37 C
6/30/14
Target: Analyze the second set of PGBR22 samples using a restriction enzyme digest and visualize the fragmentation in gel.
- calculated the various concentrations and amounts of the plasmid, enzyme buffers, restriction enzyme, and DDW.
- three different preparations of reactions: EcoRI, pvuII, and EcoRI + pvuII
- Negative control* : Regular PGBR22 plasmid without restriction enzymes
- Made gel for the gel electrophoresis
- gel ultimately not tested due to time constraints
- enzyme stop process followed to ensure
Week 4
6/27/14
- Target: Test the second sample of PGBR22 using gel electrophoresis and DNA sequencing.
- Sent second set of samples off to sequencing.
- prepared 1% Agarose Gel to see the bands and also to see the level of degradation given the origin of the samples from the backup set of flasks.
- Results:
6/26/14
- Target: Conduct a restriction enzyme digest testing the PGBR22 plasmids.
- Achieved: Due to the samples being left outside overnight, a second midi-prep was done the samples from which are hereby defined as samples of the second set.
- Ran a gel to test the samples of PGBR22 from set one using the concentration of 293.3 ng/ul. This was to test for the potential of degradation, given that the samples in the were plasmids and not proteins, they stood less chance of denaturing given the environment.
Results: The gel showed degradation, but less so than expected.
6/25/14
- Target: Send of samples of PGBR22 to sequencing and make LB and agar plates.
-
6/24/14
Target: Conducted a midi-prep to isolate DNA from PGBR22.
-after completing the midi-prep, a Nano-Drop was conducted to test the concentration of the extraction in ng/ul.
6/23/14
Target: Understand the process of analyzing a DNA sequences and restriction enzymes by using computer programs.
- prepare flasks of LB to process the next day.